Intracellular mRNA localization is a conserved mechanism for generating asymmetry in eukaryotic cells. Localization of nanos (nos) mRNA at the posterior pole of the Drosophila oocyte, and its subsequent translation, is critical for embryonic anterior-posterior axis formation and for establishment of a functional germline. Posterior localization is directed by a cis-acting signal in the nos 3'UTR comprising multiple, partially functional elements. Previous studies suggest that trans-acting factors bind to this localization signal to package nos RNA into a ribonucleoprotein (RNP) complex that permits localization. However, only one nos localization factor, the Drosophila heterogeneous ribonucleoprotein (hnRNP) M homolog, Rumpelstiltskin (Rump), has been identified thus far. Furthermore, nos localization must be tightly coupled with translational control to ensure posterior expression of Nos protein. This is achieved by another cis- acting sequence that contains binding sites for ovarian and embryonic translational repressers. Together, these repressers prevent expression of unlocalized nos mRNA. It is currently unknown how nos mRNA is coordinately regulated by localization factors and translational repressers. The goal of this work is to determine how mRNA localization signals are recognized by components of the cellular localization machinery and how the interaction of RNA-binding proteins with these signals direct RNA localization. Preliminary results identify a role for a second protein, Lost, in nos localization, and establish both genetic and biochemical interactions between Lost and Rump. These results prompt further investigations that will determine if Lost acts indirectly or directly to regulate nos localization, and will evaluate the interaction of Lost with Rump and potentially, translational control factors. Eliminating Lost and Rump does not completely abolish nos localization, likely due to functional redundancy in the nos localization signal. This result motivates the second aim of this proposal, to identify additional factors that interact with the nos mRNA localization signal and contribute to the regulation of mRNA localization and translation. Together, these experiments will allow us to determine how mRNAs are packaged for intracellular localization and how RNA localization and translational control are coordinately regulated. Abnormal expression of the proteins that control embryonic development is associated with a number of diseases. By understanding the mechanisms by which cells regulate gene expression during development, the proposed work will provide insight as to how defects in normal processes lead to diseases such as cancer.